The ATP-binding cassette transporter ABCA3 mediates choline-phospholipids uptake into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have previously shown that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease. E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes, as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, while those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[³²P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pediatric interstitial lung disease due to ABCA3 mutation.